Helsinki University Biomedical Dissertations No. 150
Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)
Elina Heliövaara, MD
Faculty of Medicine Department of Medical Genetics Genome-Scale Biology Research Program
Haartman Institute University of Helsinki
Finland
Academic dissertation
To be publicly discussed, with the permission of the Faculty of Medicine, University of Helsinki, in the Small Lecture Hall of Haartman Institute, Haartmaninkatu 3, Helsinki,
on May 6th 2011 at noon.
Helsinki 2011
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Supervised by Academy Professor Lauri A. Aaltonen, MD, PhD Department of Medical Genetics
Genome-Scale Biology Research Program Haartman Institute, University of Helsinki Helsinki, Finland
Docent Auli Karhu, PhD
Department of Medical Genetics
Genome-Scale Biology Research Program Haartman Institute, University of Helsinki Helsinki, Finland
Reviewed by Docent Pia Jaatinen, MD, PhD
School of Medicine, University of Tampere and Department of Internal Medicine Tampere University Hospital
Tampere, Finland
Docent Kirmo Wartiovaara, MD, PhD Developmental Biology
Institute of Biotechnology University of Helsinki Helsinki, Finland
Official opponent Professor Leo Niskanen, MD, PhD Department of Internal Medicine
Central Hospital of Central Finland, Jyväskylä
and University of Eastern Finland, Faculty of Health Sciences, Kuopio Campus, Finland
ISBN 978-952-10-6937-6 (paperback) ISBN 978-952-10-6938-3 (PDF) ISSN 1457-8433
http://ethesis.helsinki.fi Helsinki University Print Helsinki 2011
3 Table of Contents
List of Original Publications ... 6
Abbreviations ... 7
Abstract... 8
Review of the Literature ... 10
1. The genome and tumorigenesis ... 10
1.1 Oncogenes ... 11
1.2 Tumor suppressor genes ... 12
1.3 Genetic predisposition to tumorigenesis... 12
2. The pituitary gland ... 13
3. Pituitary adenomas... 15
3.1 Benign adenomas of the anterior lobe ... 15
3.2 Incidence and prevalence... 15
3.3 Tumor classification ... 16
3.4 Clinical features and diagnosis ... 16
3.5 Treatment and management ... 18
4. Genetics of pituitary adenomas ... 19
4.1 Sporadic pituitary adenomas ... 20
4.1.1 GNAS/gsp oncogene ... 20
4.1.2 Other features of sporadic adenomas... 20
4.2 Familial pituitary adenomas ... 21
4.2.1 Multiple endocrine neoplasia type 1 (MEN1) ... 21
4.2.2 MEN4 (MEN1-like syndrome) ... 22
4.2.3 Carney complex (CNC) ... 23
4.2.4 Pituitary adenoma predisposition (PAP)... 23
4.2.5 Isolated familial somatotropinoma (IFS) ... 25
4.2.6 Familial isolated pituitary adenoma (FIPA)... 26
5. Molecular function of the AIP protein ... 26
5.1 Features and function of AIP ... 26
5.2 AIP-related cellular pathways ... 27
5.2.1 Role of AIP in the xenobiotic response ... 27
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5.2.2 AIP in the regulation of cAMP ... 29
5.2.3 Interaction of AIP with RET and survivin ... 30
5.2.4 Other cellular interactions of AIP ... 30
Aims of the Study... 32
Subjects and Methods ... 33
1. Subjects (I, II, IV, V) ... 33
1.1 Familial pituitary adenoma patients ... 33
1.2 Assessing patient characteristics of PAP ... 33
1.3 Human pituitary adenoma samples ... 34
1.4 Healthy controls ... 35
2. MLPA assay and validation of results (I)... 35
3. Immunohistochemistry of pituitary adenomas (II, III, V) ... 35
4. Cell culture studies (II) ... 36
4.1 Aip silencing and cell proliferation assay ... 36
4.2 Western blot analyses ... 37
5. Statistical analysis (II, III, IV, V) ... 37
6. The Aip mouse model (III) ... 38
6.1 Generation of Aip mutant mice ... 38
6.2 Collection and staining of tissues ... 38
6.3 LOH analysis ... 38
6.4 Igf-1 expression analysis ... 39
7. RET sequencing and Enhancer Element Locator (EEL) analysis (V) ... 39
8. Ethical issues (I, II, III, IV, V) ... 40
Results ... 41
1. Identification of large genomic deletions in AIP (I) ... 41
1.1 Exon 2 deletion ... 41
1.2 Exon 1 and 2 deletion ... 42
2. Elucidating the expression of AIP-related proteins in pituitary adenomas (II) ... 42
2.1 Immunohistochemistry results ... 42
2.2 ARNT expression in cell lines ... 43
2.3 Cell proliferation ... 43
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3. Creating Aip mutant mice prone to pituitary adenomas (III)... 44
3.1 Phenotype and tumor spectrum of Aip mice ... 44
3.2 Loss of Aip in pituitary tumors ... 45
3.3 Proliferation index of tumors ... 45
3.4 Igf-1 expression in mice with Aip-deficient somatotropinomas... 45
3.5 Estrogen receptor expressions in Aip-deficient and -proficient tumors ... 46
3.6 ARNT/ARNT2 imbalance ... 47
4. Assessing clinical characteristics of PAP (IV) ... 47
4.1 PAP patient demographics ... 47
4.2 Characteristics of AIP mutation positive pituitary adenomas ... 47
4.3 Response to therapy ... 48
5. Evaluating the role of RET in familial pituitary adenomas (V) ... 49
5.1 RET heterozygous changes ... 49
5.2 EEL analysis ... 50
5.3 RET immunohistochemistry ... 51
Discussion ... 52
1. Large genomic AIP deletions account for a subset of AIP mutations (I) ... 52
2. ARNT is underexpressed in AIP mutation positive pituitary adenomas (II) ... 53
2.1 Implications of ARNT in other cellular pathways ... 54
2.2 ARNT underexpression requires a pituitary tumor environment ... 55
3. Aip heterozygous mice are extremely prone to pituitary adenomas (III) ... 55
3.1 Aberrant ARNT/ARNT2 expression in Aip-deficient pituitary adenomas ... 57
3.2 Other characteristics of Aip-deficient and -proficient pituitary adenomas ... 58
4. AIP mutation positive patients display an aggressive disease phenotype (IV)... 59
5. No evidence of RET mutations in familial pituitary adenoma patients (V) ... 62
5.1 EEL results provide data on regulation of RET transcription ... 62
5.2 RET underexpression in AIP mutation positive somatotropinomas... 63
Conclusions and Future Prospects ... 64
Acknowledgements ... 66
References ... 68
6 List of Original Publications
This thesis is based on the following five original articles. They will be referred to in the text by the Roman numerals I-V.
I Georgitsi M, Heliövaara E, Paschke R, Kumar AV, Tischkowitz M, Vierimaa O, Salmela P, Sane T, De Menis E, Cannavò S, Gündogdu S, Lucassen A, Izatt L, Aylwin S, Bano G, Hodgson S, Koch CA, Karhu A, Aaltonen LA. Large genomic deletions in aryl hydrocarbon receptor interacting protein (AIP) gene in pituitary adenoma predisposition. J Clin Endocrinol Metab 2008, 93(10):4146-4151
II Heliövaara E*, Raitila A*, Launonen V, Paetau A, Arola J, Lehtonen H, Sane T, Weil R, Vierimaa O, Salmela P, Tuppurainen K, Mäkinen M, Aaltonen LA, Karhu A. The expression of AIP-related molecules in elucidation of cellular pathways in pituitary adenomas. Am J Pathol 2009, 175(6):2501-2507
III Raitila A, Lehtonen HJ, Arola J, Heliövaara E, Ahlsten M, Georgitsi M, Jalanko A, Paetau A, Aaltonen LA, Karhu A. Mice with Inactivation of Aryl Hydrocarbon Receptor Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with aberrant ARNT Expression. Am J Pathol 2010, 177(4):1969-1976 IV Daly AF*, Tichomirowa MA*, Petrossians P*, Heliövaara E, Jaffrain-Rea ML,
Barlier A, Naves LA, Ebeling T, Karhu A, Raappana A, Cazabat L, De Menis E, Montañana CF, Raverot G, Weil RJ, Sane T, Maiter D, Neggers S, Yaneva M, Tabarin A, Verrua E, Eloranta E, Murat A, Vierimaa O, Salmela PI, Emy P, Toledo RA, Sabaté MI, Villa C, Popelier M, Salvatori R, Jennings J, Ferrandez Longás A, Labarta Aizpún JI, Georgitsi M, Paschke R, Ronchi C, Välimäki M, Saloranta C, De Herder W, Cozzi R, Guitelman M, Magri F, Lagonigro MS, Halaby G, Corman V, Hagelstein MT, Vanbellinghen JF, Barra GB, Gimenez-Roqueplo AP, Cameron FJ, Borson-Chazot F, Holdaway I, Toledo SP, Stalla GK, Spada A, Zacharieva S, Bertherat J, Brue T, Bours V, Chanson P, Aaltonen LA, Beckers A. Clinical characteristics and therapeutic responses in patients with germ-line AIP mutations and pituitary adenomas: an international collaborative study. J Clin Endocrinol Metab 2010, 95(11):E373-383 V Heliövaara E, Tuupanen S, Ahlsten M, Hodgson S, de Menis E, Kuismin O, Izatt L,
McKinlay Gardner RJ, Gündogdu S, Lucassen A, Arola J, Tuomisto A, Mäkinen M, Karhu A, Aaltonen LA.No evidence of RET germline mutations in familial pituitary adenoma. J Mol Endocrinol 2011, 46(1):1-8
*Equal contribution
The original publications are reproduced with the permission of the copyright holders.
7 Abbreviations
aa amino acid
ACTH adrenocorticotropin
AIP aryl hydrocarbon receptor interacting protein (or ARA9, XAP2)
AHR aryl hydrocarbon receptor
ARA9 aryl hydrocarbon receptor-associated protein-9 (or AIP, XAP2)
ARNT aryl hydrocarbon receptor nuclear translocator (or HIF1- ) ARNT2 aryl hydrocarbon receptor nuclear
translocator 2 aSU alpha subunit
bp base pair
BRCA1 breast and ovarian cancer 1 BRCA2 breast and ovarian cancer 2
bSU beta subunit
cAMP cyclic adenosine monophosphate CD34 cluster of differentiation 34
CDKN1B cycline-dependent kinase inhibitor 1B cDNA complementary deoxyribonucleic acid CEPH Centre d’Étude du Polymorphisme Humain
CNC Carney complex
CYP cytochrome P450
DAB 3,3’-diaminodenzidine DNA deoxyribonucleic acid DRE dioxin response element
E (mouse) embryonic day
EBNA-3 Epstein-Barr virus encoded nuclear antigen- 3
EEL enhancer element locator
ER estrogen receptor
ER estrogen receptor
ES cells embryonic stem cells
ETV ETS variant gene 6
FGFR fibroblast growth factor receptor FIPA familial isolated pituitary adenoma FKBP FK605 binding protein
FSH follicle-stimulating hormone G13 G protein subtype 13
GDNF glial cell line-derived neurotrophic factor
GH growth hormone
GHRH growth hormone-releasing hormone GLUT1 glucose transporter 1
GNRH gonadotropin-releasing hormone
GNAS guanine nucleotide-binding protein alpha stimulating activity polypeptide
GR glucocorticoid receptor HBV hepatitis B virus
HE haematoxylin-eosin
HEK293 human embryonic kidney 293 cells
HET heterozygote
HIF hypoxia inducible factor
HIF1- hypoxia inducible factor 1, alpha subunit HIF1- hypoxia inducible factor 1, beta subunit (or
ARNT)
HLRCC hereditary leiomyomatosis and renal cell cancer
HRE hypoxia response element Hsc70 heat-shock cognate protein 70 HSP90 heat-shock protein 90
iASPP inhibitor of apoptosis stimulating protein of p53
IFS isolated familial somatotropinoma IGF-I insulin-like growth factor 1
IHC immunohistochemistry
IVS intronic variable sequence
iXRE inhibitory xenobiotic response element
kb kilobase
kDa kilodalton
LH luteinizing hormone
LOH loss of heterozygosity
MAS McCune-Albright syndrome
MCS multi-species conserved sequence MEF mouse embryonic fibroblast MEN1 multiple endocrine neoplasia type 1 MEN2A multiple endocrine neoplasia type 2A MEN2B multiple endocrine neoplasia type 2B MEN4 multiple endocrine neoplasia type 4 (or
MEN1-like syndrome) MENX MEN-like syndrome in the rat MLPA multiplex ligation-dependent probe
amplification
MRI magnetic resonance imaging mRNA messenger ribonucleic acid NFPA non-functioning pituitary adenoma OGTT oral glucose tolerance test PAP pituitary adenoma predisposition PCR polymerase chain reaction PDE2A phosphodiesterase 2A PDE4A5 phosphodiesterase 4A5
PI proliferation index
Pit-1 pituitary transcription factor-1
PKA protein kinase A
PPAR- peroxisome proliferation-activated receptor alpha
PRKAR1A protein kinase A regulatory subunit 1 alpha
PRL prolactin
PTTG pituitary tumor transforming gene qPCR quantitative polymerase chain reaction RET rearranged during transfection RSUME RWD-containing sumoylation enhancer
S serum
siRNA small interfering ribonucleic acid SNP single nucleotide polymorphism SRF serum response factor
SSA somatostatin analog
T3 triiodothyronine
T4 thyroxine
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TOMM20 translocase of the outer membrane of
mitochondria 20 TPR tetratricopeptide repeat TR 1 thyroid hormone receptor beta 1 TRH thyrotropin-releasing hormone TSH thyroid-stimulating hormone UFC urinary free cortisol UTR untranslated region
VEGF vascular endothelial growth factor
WT wildtype
XAP2 hepatitis B virus x-associated protein 2 (or AIP, ARA9)
In addition, standard one-letter codes are used to denote amino acids and bases.
8 Abstract
Pituitary adenomas are common intracranial neoplasms that generally arise sporadically. However, a small minority occurs in a familial setting, often as aggressive and difficult-to-treat adenomas in patients who are relatively young. Familial syndromes with phenotypes including pituitary adenomas include multiple endocrine neoplasia type 1 (MEN1), Carney complex and MEN4. Recently, a fourth gene underlying pituitary adenomas was discovered in Northern Finland in a cluster of familial acromegaly. Heterozygous mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene caused this condition, designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of pituitary adenomas, and patients often lack a strong familial background of adenomas.
AIP mutation positive (AIPmut+) patients are often young at disease onset and have mostly growth hormone (GH) secreting adenomas. Loss of heterozygosity of AIP in tumors and functional evidence suggest that AIP is a tumor suppressor gene.
Elucidation of the molecular mechanisms of PAP is a requirement for better understanding of the detailed genesis of these pituitary adenomas. Moreover, clarification of the clinical characteristics of PAP may be beneficial in establishing genetic testing protocols to recognize individuals at risk for developing tumors and to improve patients’ clinical outcome. Development of novel treatment regimes relies on detailed knowledge of tumorigenesis. This thesis work aims to clarify the molecular and clinical characteristics of PAP.
Applying the multiplex ligation-dependent probe amplification (MLPA) assay, we searched for large genomic AIP deletions in apparently AIP mutation negative (AIPmut-) familial pituitary adenoma patients. For the first time, genomic AIP deletions were found in two families, suggesting that this mutation type accounts for a subset of PAP. Therefore, MLPA could be considered in cases with a phenotype indicative of PAP but when no AIP mutations are found with conventional sequencing.
To clarify molecular mechanisms of AIP-mediated tumorigenesis, we elucidated the expression of AIP-related molecules in AIPmut+ and AIPmut- pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) protein was reduced in AIPmut+ pituitary adenomas, whereas the nuclear expression of aryl hydrocarbon receptor (AHR) was somewhat increased. This result was endorsed by underexpression of ARNT in an Aip knockdown rat mammosomatotroph cell line. These results suggest that ARNT, AHR or both may play a role in AIP-related tumorigenesis, possibly via pathways involving phosphodiesterases and cyclic adenosine monophosphate.
We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. Heterozygous Aip mutations conferred complete penetrance of pituitary adenomas in these mice, and the vast majority of adenomas were GH-secreting. Thus, the tumor phenotype of the Aip mouse is similar to that in human PAP patients. As in the study on human tumors, aberrant ARNT, but also ARNT2 expression, was evident in mouse pituitary adenomas that were Aip-deficient. Our results suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. Furthermore, this disease model is an
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excellent tool in further elucidation of the molecular mechanisms of pituitary tumorigenesis, and it also has potential in developing therapeutic approaches.
We studied the clinical characteristics and the response to therapy of AIPmut+ pituitary adenoma patients, with sporadic acromegaly patients as a control population. AIPmut+ adenomas conferred an aggressive disease phenotype with young age at disease onset. AIPmut+ adenomas were most often large, expansive and invasive at diagnosis. Patients were predominantly male, and GH-secreting adenomas appeared in nearly 80%. AIPmut+ adenomas also seemed to have many difficult-to-treat clinical characteristics. The aggressive nature of AIPmut+ adenomas is further supported by increased expression of the Ki-67 proliferation marker in mouse pituitary adenomas that are Aip-deficient. We conclude that the improvement in treatment outcomes for PAP patients would require efficient identification of AIPmut+ patients, as well as earlier diagnosis of the pituitary adenomas.
The possible role of the rearranged during transfection (RET) proto-oncogene in tumorigenesis of familial AIPmut- pituitary adenomas was evaluated. Five novel germline heterozygous RET variants were found in the patients; however, none of these could be considered causative of pituitary tumorigenesis. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIPmut+ pituitary adenomas – an observation that merits further investigation.
10 Review of the Literature
1. The genome and tumorigenesis
The human body is composed of approximately 3 x 1013 cells, nearly all of which contain the same genetic material. This material resides in the nucleus as chromosomes that contain the deoxyribonucleic acid (DNA) sequence, which encodes the ~22 000 genes of the genome. In addition, a small fraction of DNA is contained within the mitochondria. Genes encoded by the DNA sequence constitute the templates for amino acid (aa) sequences of proteins that carry out the genes’ purposes in the cells; the genotype creates the phenotype of an individual through proteins. Gene expression is stringently regulated in time and space. An important example of this is the regulation of the expression of genes involved in cell division of somatic cells. Proper tissue architecture is maintained by appropriate proportions of constituent cell types, replacement of missing cells and discarding of unneeded cells. This process of normal growth involves a delicate balance between growth factors and growth-inhibitory factors in the surroundings of cells (Weinberg 2007 p.9, 19, 43, 121).
Deviations from normal growth can involve hyperplasia, designating an excessive numbers of cells;
metaplasia, when certain cells are displaced by cells of another type that are normally not encountered in that site; dysplasia, when cells have reached a cytologically abnormal stage; and ultimately neoplastic and metastatic lesions. The genesis of tumors results from the abnormal proliferation of normal cells, which is accompanied by the accumulation of genetic defects in these cells (Weinberg 2007 p.36-39, 43). A succession of genetic changes confers a growth advantage, leading to the progressive conversion of normal cells into cancer cells (Hanahan & Weinberg 2000). Since the karyotypes of cancer cells are usually abnormal, cancer can be seen as a genetic disease of somatic cells (Knudson 2002). Recent evidence suggests that cancers of distinct subtypes within an organ may be derived from different ‘cells of origin’, and that these are the cells that acquire the genetic changes that culminate in the initiation of cancer (Levy 2008, Vankelecom 2011, Visvader 2011).
Tumors are initiated by the first genetic alteration that renders a fitness advantage to a cell, and tumor progression is the multi-step evolution of a normal cell into a tumor cell (Weinberg 2007 p.G20, Bozic et al. 2010). The mutations that are essential in the initiation of tumorigenesis are known as
“drivers” and they confer a growth advantage, causing the positive selection of the cell in which they occur. However, the majority of somatic mutations are “passengers”, which are expected to be biologically neutral since they do not confer a growth advantage to the cell in which they occur and they are not causative of oncogenesis (Greenman et al. 2007). It has been reported that, for example, in typical breast and colorectal cancers there are ~80 aa-altering mutations in the tumor DNA and that
~15 of these mutations are likely to be responsible for driving initiation, progression or maintenance of the tumor (Wood et al. 2007).
It has been postulated that cancer cells comprise six essential alterations in cell physiology that define their malignant growth. These are self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), a limitless replicative potential of cells, sustained angiogenesis, and tissue invasion and metastasis (Hanahan & Weinberg 2000). Also genomic instability, for example resulting from mutations in DNA repair genes, has recently been
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suggested to be a hallmark of cancer (Negrini et al. 2010, Hanahan & Weinberg 2011). Emerging views also suggest that the genetic changes in cancer cells are not sufficient and that tumor progression is dependent on ancillary processes provided by the tumor environment but not necessarily cancerous themselves, such as inflammation and a shift in cellular metabolism (Rakoff- Nahoum 2006, Tennant et al. 2009, Hanahan & Weinberg 2011).
In contrast to malignant cancer, benign tumors are by definition confined to a specific site of a tissue and give no evidence of invading adjacent tissues (Weinberg 2007 p.G2). They are composed of well- differentiated cells that closely resemble their normal counterparts, and their rate of growth is usually slow (Kumar et al. 2003 p.168-173). However, benign tumors can cause significant morbidity and even mortality, for example pituitary adenomas can do so by compressing critical brain structures (Elston et al. 2009).
1.1 Oncogenes
Tumor cells can exhibit abnormal activation of certain normal genes to promote tumorigenesis. These genes are called oncogenes and one mutated allele of such a gene is sufficient to confer a selective growth advantage to the cell. Oncogene activation can be caused by chromosomal translocations, gene amplifications or intragenic mutations affecting crucial residues that regulate the activity of the gene product (Vogelstein & Kinzler 2004). Translocations and intragenic mutations can occur either as initiating effects of tumorigenesis or during tumor progression, whereas gene amplification usually occurs during tumor progression (Croce 2008). Oncogenes encode proteins that can control cell proliferation, apoptosis or both. The products of these oncogenes can be classified into six groups of molecules: transcription factors, chromatin remodelers, growth factors, growth factor receptors, signal transducers and apoptosis regulators. Known oncogenes of the six classes include v-myc (transcription factor), ALL1 (chromatin remodeler), KS3 (growth factor), rearranged during transfection (RET) proto-oncogene (growth factor receptor), K-RAS (signal transducer) and BCL2 (apoptosis regulator) (Croce 2008, Table 1).
Table 1. Oncogenes and tumor suppressor genes, data from Bronner et al. 1994, Kinzler & Vogelstein 1998, Soussi 2000, Weber et al. 2006 and Croce 2008.
Gene Mechanism in tumorigenesis
Oncogene Activating mechanism
v-myc deregulated activity of transcription factor
ALL1 chromatin remodelling
KS3 constitutive production of growth factor RET constitutive action of growth factor receptor
K-RAS signal transduction
BCL2 inhibition of apoptosis
Tumor suppressor gene Inactivating mechanism
p53 evading apoptosis (gatekeeper)
RB1 uncontrolled proliferation (gatekeeper) MLH1 genome instability (caretaker)
BRCA1 genome instability (caretaker)
POLD1 generating unstable stroma (landscaper)
12 1.2 Tumor suppressor genes
Tumor suppressor genes are antigrowth genes whose involvement in tumor formation occurs when these genes are inactivated or lost (Weinberg 2007 p.209-210). A model for tumor suppressor gene associated cancer development was proposed in 1971, when Alfred Knudson published his “two-hit”
hypothesis based on epidemiological studies on retinoblastoma patients. He suggested that a mutational event must occur in both alleles of a tumor suppressor gene for the affected cell to acquire a growth advantage (Knudson 1971).
Mutations in tumor suppressor genes have the opposite effect to oncogene mutations, since they reduce the activity of the gene product. Such inactivation can arise from amino acid changes at residues that are essential for the activity of the gene product, from mutations that result in a truncated protein, from gene deletions or insertions, or from epigenetic silencing (Vogelstein & Kinzler 2004).
Interestingly, some tumor suppressor genes exert a selective advantage on the cell even when only one allele is inactivated and the other remains functional, a situation that is called haploinsufficiency (Santarosa & Ashworth 2004). However, inactivation of both alleles of a tumor suppressor gene is generally required to confer a selective advantage to the cell. This situation often arises through an intragenic mutation in one allele, coupled with a deletion of the other allele via a gross chromosomal event (Knudson 2002). Generally, this phenomenon where the second allele is lost is called loss of heterozygosity (LOH) (Weinberg 2007 p.219-224).
Tumor suppressor genes can have functions over the control of cellular proliferation directly acting as
“gatekeepers”, or they can function in maintaining the integrity of the genome as “caretakers”.
Inactivation of a caretaker gene does not promote tumor initiation directly, but does so indirectly by leading to genetic instability which results in increased mutation rates of all genes including gatekeepers (Kinzler & Vogelstein 1997). Classical tumor suppressor genes from these groups include p53 (gatekeeper), MLH1 (caretaker) and breast and ovarian cancer 1 and 2 (BRCA1 and BRCA2) (caretakers) (Bronner et al. 1994, Kinzler & Vogelstein 1998, Soussi 2000, Table 1). Interestingly, some mutant forms of p53 can also act as oncogenes (Harris & Hollstein 1993). A third group of tumor suppressor genes are the “landscapers”. It is postulated that alterations in landscaper genes can cause proliferation of stromal cells. This genetically unstable stroma results in an abnormal microenvironment that may promote neoplastic transformation of associated epithelial cells (Kinzler
& Vogelstein 1997, Kinzler & Vogelstein 1998, Weber et al. 2006, Table 1).
1.3 Genetic predisposition to tumorigenesis
Inherited predisposition is known for virtually every type of human cancer (Knudson 2002). The predisposed individual carries a defect in the germline, such as a mutated allele of a gene, which predisposes him or her to the formation of tumors. This predisposition can also be transferred to the offspring of the individual. Usually a certain amount of loss of germline mutations occurs in every generation, for example if the condition produces mortality before the end of the age of reproduction.
However, mutational equilibrium can be attained by a low rate of occurrence of new germline mutations in the population (Knudson 2002, Weinberg 2007 p.43, 224-226).
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There are currently about 100 genes known to cause Mendelian-inherited cancer syndromes (Cazier &
Tomlinson 2010). Clinical characteristics of inherited cancer syndromes include, among others, a positive family history of cancer, a typical inheritance pattern of tumors, multiple primary tumors and early age of onset (D’Orazio 2010). It has been argued that these syndromes affect about 1 % of cancer patients, and in children it is estimated that 5 to 10% of cancer can be explained by a certain genetic mutation (Fearon 1997, D’Orazio 2010). Inherited predisposing mutations in oncogenes have been identified in several well-established syndromes that cause dominant heredity of cancer, such as RET mutations causing thyroid, parathyroid and adrenal tumors in multiple endocrine neoplasia type 2A (MEN2A) (Mulligan et al. 1993, Salmela and Ebeling in Välimäki et al. 2009 p.474-480, Table 1). Inherited mutations in tumor suppressor genes can also confer a dominant pattern of heredity.
These include for example RB1 mutations causing retinoblastomas and FH mutations causing hereditary leiomyomatosis and renal cell cancer (HLRCC) (Knudson 1971, Tomlinson et al. 2002, Table 1). Hereditary cancer predisposition can also be caused by mutations in stability genes (also called caretakers), such as BRCA1 and BRCA2, resulting in dominant inheritance of breast and ovarian cancer, and FANCA mutations, causing recessive inheritance of leukemia (Butturini et al.
1994, Futreal et al. 1994, Miki et al. 1994, Wooster et al. 1995, Vogelstein & Kinzler 2004). The known Mendelian-inherited cancer syndromes explain only a minor part of the familial clustering of cancers. Thus, in most cases, the increased familial relative risk of cancer must involve several risk alleles with low or moderate penetrance (Cazier & Tomlinson 2010). It has indeed been estimated in twin studies that hereditary factors could significantly contribute to prostate cancer (42% of risk may be explained by heritable factors), colorectal cancer (35%) and breast cancer (27%) (Lichtenstein et al. 2000).
It is known that a certain germline mutation does not suffice for carcinogenesis, but subsequent somatic mutations are required. These can be caused by various environmental factors, such as ionizing radiation, dietary factors and consumption of tobacco. These factors can also affect the penetrance of cancer, i.e. the proportion of genetically predisposed individuals that ultimately develop tumors (Knudson 2002, Weinberg 2007 p.47, Cazier & Tomlinson 2010).
2. The pituitary gland
The pituitary gland is a crucial part of the endocrine system. It co-ordinates the body’s internal physiology, regulates its development throughout life and helps it to adapt to change in the external environmental by secreting hormones that act on their target tissues (Brook & Marshall 2001 p.34).
The pituitary is composed of three lobes: the anterior lobe (adenohypophysis; contains the pars distalis and pars tuberalis) is mainly glandular tissue; the posterior lobe (neurohypophysis; contains the pars nervosa and infundibulum) is neural tissue that stores oxytocin and antidiuretic hormone; and between them the intermediate lobe (pars intermedia), which is atrophic in humans (Sane in Välimäki et al. 2009 p.76-77). The pituitary lies in a bony cavity, the sella turcica of the sphenoid bone. It is connected to the overlying hypothalamus by a stalk that carries a system of portal veins from the hypothalamus to the pituitary, as well as axons to the neurohypophysis (Brook & Marshall 2001 p.35- 37).
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During embryogenesis, the pituitary emerges from two distinct ectodermal components. One of these is the Ratkhe’s pouch, a dorsal outgrowth of the buccal cavity, which forms the anterior pituitary. The second is a downgrowth of neuroectoderm from the floor of the third ventricle, which develops into the pituitary stalk and the posterior pituitary (Brook & Marshall 2001 p.35). The distinct cell types of the anterior pituitary arise from a pool of self-renewing and proliferating progenitor cells present in the epithelium of Ratkhe’s pouch (Vankelecom 2010). Mitotic activity of the adult pituitary is seen in 1-2% of cells that are active oligopotent stem cells. They undergo mitoses at a steady rate that gradually decreases with age (Levy 2008). Interestingly, the adult pituitary seems to retain plasticity and is able to flexibly remodel its hormone-producing cell compartment in response to changing endocrine demands, for example during pregnancy and puberty. Recently, plausible candidates for stem cells of the adult pituitary have been proposed, that express stem cell-associated markers and signaling factors, and display multipotency and a niche-like organization (Vankelecom 2010).
The anterior pituitary contains five populations of secretory cells that secrete by exocytosis the six pituitary hormones into the bloodstream. Growth hormone (GH) is secreted by somatotrophs, prolactin (PRL) by lactotrophs, thyroid-stimulating hormone (TSH) by thyrotrophs, adrenocorticotropic hormone (ACTH) by corticotrophs, and both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are secreted by gonadotrophs. FSH and LH are dimeric proteins that share a common alpha subunit (aSU), but their beta subunits (bSU) are unique and derived from different genes encoding distinct proteins (Brook & Marshall 2001 p.38, Ooi et al. 2004, Bernard et al. 2010, Figure 1).
Figure 1. Pituitary gland. A schematic presentation of the cells in the anterior pituitary, the hormones they secrete, and the target tissues and main effects of these hormones. Modified from Ooi et al. 2004 with the permission from Elsevier.
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The anterior pituitary lies under the stringent control of the hypothalamus, which controls the release of pituitary hormones by releasing hypothalamic hormones such as growth hormone-releasing hormone (GHRH) and gonadotropin-releasing hormone (GNRH) to the hypophysial portal vasculature. In addition, some neuromessengers can directly control the release of pituitary hormones;
for example dopamine is the main inhibitor of PRL secretion. Also feedback effects of systemic circulating hormones and cytokines acting in a paracrine or autocrine fashion regulate the secretion of pituitary hormones. (Crowley 1999, Brook & Marshall 2001 p. 38-40, Haedo et al. 2009, Sane in Välimäki et al. 2009 p.69, 75-76).
3. Pituitary adenomas
3.1 Benign adenomas of the anterior lobe
Pituitary adenomas account for about 15% of intracranial neoplasms. They can arise from any cell type(s) of the anterior pituitary and accordingly present a variety of clinical manifestations based on their size, location and function. Frequently encountered clinical manifestations relate to excessive hormone secretion of the tumor, hormone deficits of the pituitary hormones (i.e. hypopituitarism) and expansion of the tumor mass. However, pituitary adenomas can also be asymptomatic. Although some adenomas are invasive, the vast majority of them are considered as histologically benign lesions, and they metastasize exceedingly rarely. Recent advances in molecular biology, immunocytochemistry and imaging, and the introduction of new treatment options have improved the knowledge of these adenomas and their management (Arafah & Nasrallah 2001, Melmed 2003, Karhu & Aaltonen 2007, Sane in Välimäki et al. 2009 p.98-126).
3.2 Incidence and prevalence
According to data obtained from autopsy and radiological imaging series, pituitary adenomas occur very commonly in the general population. Most of these tumors are found incidentally and present no obvious clinical impact (Daly et al. 2009). In an early study on pituitary adenoma prevalence, adenomas were found in nearly one in every four autopsy cases (Costello 1936). In a recent systematic review, the overall prevalence of pituitary adenomas was found to be 16.7% (14.4% based on autopsy studies and 22.5% based on radiological studies) (Ezzat et al. 2004). Incidence rates of pituitary adenomas generally increase with age and are higher in women in early life and higher in men in later life. The difference in incidence rates between sexes could be due to their different symptomatology, such as earlier and more noticeable symptoms of hyperprolactinemia in women (e.g.
amenorrea and galactorrhea). Men are on average diagnosed with larger tumors than women since their diagnosis may be delayed, giving the tumor a chance to grow larger before clinical detection (Ciccarelli et al. 2005, McDowell et al. 2010). Also race may affect the incidence of pituitary adenomas, for example incidence rates for women of African descent are about three times as high as for Caucasian women (Heshmat et al. 1976). The overall incidence rate of pituitary adenomas in the United States has been noted to be 2.7 cases per 100 000 patient years in 2004-2007 (McDowell et al.
2010). In contrast, incidences noted in England have been lower, only 0.75 cases per 100 000 patient years in 1999-2003. In England, there has been a pattern of initial increase (0.91 per 100 000 in 1989- 1993) followed by stabilization, and this change in incidence has mainly been seen in the elderly age group (Arora et al. 2010). In Northern Finland, overall incidence of pituitary adenomas in 1992-2007
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was 4.0 cases per 100 000 patient years, with a gender-specific incidence of 2.2 per 100 000 in males and 5.9 per 100 000 in females (Raappana et al. 2010).
3.3 Tumor classification
Pituitary adenomas are traditionally classified as microadenomas if they are <10mm in diameter and located totally within the sella turcica, in contrast to macroadenomas that are >10mm in diameter and can be totally intrasellar, but are often associated with extrasellar extension. In addition, giant adenomas have been defined as extending >40mm from the midpoint of the jugum sphenoidale or extension to within 6mm of the foramen of Monro (Majós et al. 1998, Arafah & Nasrallah 2001).
However, the general classification of pituitary adenomas is based on characteristics of hormone staining, electron microscopic changes, clinical signs and symptoms. Hereby, adenomas are classified as prolactinomas, somatotropinomas, adrenocorticotropinomas, gonadotropinomas, thyrotropinomas, null-cell adenomas and oncocytomas (Arafah & Nasrallah 2001, Table 2). The World Health Organization’s complete clinicopathological five-tier scheme for pituitary adenoma classification includes assessment of endocrine activity, imaging, operative findings, histology, immunocytochemistry and ultrastructure of the pituitary adenoma (Kovacs et al. 1996).
Table 2. Classification and characteristics of pituitary adenoma types. Modified from Arafah &
Nasrallah with the permission from the Society for Endocrinology, data also from Sane in Välimäki et al. 2009 p.100 and Melmed 2003.
Tumor type Secretion Prevalence Symptoms of hormone secretion Laboratory diagnosis Prolactinoma PRL 27-45% hypogonadism, galactorrhea S-PRL
Somatotropinoma GH 15-20% acromegaly, gigantism OGTT, S-IGF-I , S-GH Adrenocorticotropinoma ACTH 9-12% Cushing's disease Dexamethasone test,
24hUFC Gonadotropinoma LH, FSH,
a/bSU
9-15% none, hypergonadism or hypogonadism
S-FSH , S-LH , S-a/bSU
Thyrotropinoma TSH 1-2% hyperthyroidism TRH test, T , T , TSH
NFPA¹ none 5-25% none none
PRL, prolactin; GH, growth hormone; ACTH, adrenocorticotropic hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; a/bSU, alpha/beta subunit; TSH, thyroid-stimulating hormone; NFPA, non- functioning pituitary adenoma; OGTT, oral glucose tolerance test; S-IGF-I, serum insulin-like growth factor 1; TRH, thyrotropin-releasing hormone; T , triiodothyronine T , thyroxine; UFC, urinary free cortisol
¹ Including null-cell adenomas and oncocytomas
3.4 Clinical features and diagnosis
The majority of pituitary adenomas are asymptomatic and have no clinical impact (Daly et al. 2009).
However, based on population studies the prevalence of clinically relevant adenomas is higher than previously thought at about 94 in 100 000 patients in the general population (Clayton 1999, Daly et al.
2006b). In addition to hormone hypersecretion, there are two main mechanisms by which pituitary adenomas can cause clinical symptoms. These are compressive pituitary failure, causing e.g.
hypogonadism, thyroid failure or adrenal failure, and central mass effects, causing e.g. visual field disturbances, headaches and cranial nerve palsies (Melmed 2003).
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Prolactinomas are the most common type of pituitary adenomas, accounting for 27-45% of all pituitary adenomas (Arafah & Nasrallah 2001, Sane in Välimäki et al. 2009 p.100, Table 2).
However, even higher prevalence of prolactinomas (66.2% of pituitary adenomas) has been suggested (Daly et al. 2009). Prolactinomas occur more frequently in women than men until after the fifth decade of life when their frequency equalizes between the sexes (Mindermann & Wilson 1994). The most common clinical features of prolactinomas are hypogonadism and/or galactorrhea in both males and females, and they cause amenorrhea, oligomenorrhea or infertility in females and decreased libido or diminished sexual potency in males. Due to this different symptomatology in sexes, women usually present earlier than men and often exhibit microprolactinomas at diagnosis; men present later and with a higher frequency of macroprolactinomas and attendant mass effects (Ciccarelli et al. 2005).
The diagnosis is based on symptoms, an elevated serum PRL (S-PRL) level and magnetic resonance imaging (MRI). Other causes of hyperprolactinemia should be ruled out in prolactinoma diagnostics, such as pregnancy and consumption of certain medications (Colao 2009).
GH-secreting somatotropinomas account for approximately 15-20% of pituitary adenomas and cause acromegaly in adults and gigantism if they occur in children before epiphyseal plate fusion (Arafah &
Nasrallah 2001, Keil & Stratakis 2008, Sane in Välimäki et al. 2009 p.100, Table 2). The incidence of somatotropinomas is about 3-4 cases in a million patient years (Lissett et al. 1998, Kauppinen- Mäkelin et al. 2005). About 25% of somatotropinomas also co-secrete PRL. These mixed adenomas can be dimorphous adenomas composed of GH and PLR cells. They can also be monomorphous mammosomatotroph adenomas derived from mammosomatotrophs that can secrete both GH and PRL. A third possibility is that they are derived from a more primitive acidophil stem cell, which is the progenitor of somatotrophs and lactotrophs. The clinical manifestations of acromegaly are derived from the major organ systems of the body: the musculosceletal, integumentary, gastrointestinal, cardiovascular, pulmonary, endocrine and metabolic systems. Symptoms can be subtle signs of acral overgrowth (arthralgias, jaw prognathism and frontal bone bossing), soft-tissue swelling, fasting hyperglycemia and hyperhidrosis. At the other end of the spectrum are symptoms of florid osteoarthritis, diabetes mellitus, hypertension and respiratory and cardiac failure. Before epiphyseal plate closure, excessive GH leads to linear growth acceleration and gigantism (Melmed 2006, Sane in Välimäki et al. 2009 p.113-116). The mortality rate of acromegalics is double that of the general population. However, it has been reported that mortality reverts to expected levels when there is reduction of GH to less than 1µg/l or normalization of insulin-like growth factor 1 (IGF-I), which is the prime mediator of the effects of GH on target tissues (Holdaway et al. 2004). In a survey of mortality in acromegaly in Finland, posttreatment GH less than 2.5µg/l was associated with a normal life-span (Kauppinen-Mäkelin et al. 2005). Apart from typical symptoms and imaging, the diagnosis of acromegaly can be based on the inability of the patient to suppress GH levels during an oral glucose-tolerance test (OGTT), the excessive peripheral biologic effects of GH reflected by elevations in S-IGF-I levels, as well as elevated S-GH levels (Melmed 2006, Sane in Välimäki et al. 2009 p.116- 119).
ACTH-secreting adenomas, known as adenocorticotropinomas, account for 9-12% of pituitary adenomas and are seen predominantly in females. They cause Cushing’s disease, a state of hypercortisolism caused by excess pituitary secretion of ACTH, which stimulates the secretion of cortisol by the adrenal glands. Symptoms of hypercortisolism include central obesity, easy bruising,
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proximal myopathy, striae, hypertension, hirsutism, menstrual irregularity, mood changes, poor wound healing, osteoporosis and hyperglycemia. Initial screening of suspected Cushing’s disease patients is achieved by an overnight 1-1.5mg dexamethasone suppression test where dexamethasone fails to suppress S-cortisol levels in Cushing’s patients, or a 24h urinary free cortisol (UFC) measurement, followed by pituitary MRI (Arafah & Nasrallah 2001, Sane in Välimäki et al. 2009 p.100, Table 2).
Gonadotropinomas account for 9-15% of pituitary adenomas. They can secrete LH, FSH or both, or their respective subunits aSU and bSU. However, their hormone secretion is often minimal or inefficient and the clinical behavior is thus often that of an inactive tumor (Samuels & Ridgway 1995). If present, the most common clinical presentations of gonadotropinomas are related to mechanical effects of the expanding macroadenoma, and it is common that the adenomas are large and extend beyond the sella turcica at diagnosis. In rare cases, patients may have symptoms of excessive hormone secretion, for example increased libido in men and ovarian hyperstimulation syndrome in women (Arafah & Nasrallah 2001, Table 2). Diagnosis of gonadotropinomas is based on measurements of serum hormone concentrations of intact FSH, intact LH and a/bSU, and tumor imaging with MRI (Daneshdoost et al. 1991, Young et al. 1996, Arafah & Nasrallah 2001).
Thyrotroph adenomas that secrete TSH are rare, accounting for 1-2% of pituitary adenomas. Patients often have goiter and evidence of mild hyperthyroidism, such as hyperhidrosis and increased appetite.
By the time of diagnosis, tumors are often large and have extrasellar extension, causing signs of hyperthyroidism and symptoms of mechanical compression. However, 30% of tumors also show increased secretion of GH or PRL, which can complicate the symptoms in these patients. Diagnosis of thyrotropinomas is based on increases in S-TSH and the serum thyroid hormone levels thyroxine (T4) and triiodothyronine (T3). Also a thyroid-releasing hormone (TRH) stimulation test without a rise in S-TSH indicates the possibility of a thyrotropinoma, and diagnosis should be followed with MRI scanning (Arafah & Nasrallah 2001, Roelfsema et al. 2009, Sane in Välimäki et al. 2009 p.123-124, Table 2).
Approximately 30% of pituitary adenomas are endocrinologically silent i.e. they cause no clinical symptoms related to excessive hormone secretion. These are often true non-functioning pituitary adenomas (NFPA) that include null-cell adenomas or oncocytomas. They usually present with mechanical effects of the adenoma and variable degrees of hypopituitarism, and their diagnosis is based on MRI scanning (Arafah & Nasrallah 2001, Heaney & Melmed 2004; Sane in Välimäki et al.
2009 p.125, Table 2).
3.5 Treatment and management
Goals in the treatment of pituitary tumors include controlling clinical and biochemical signs of excessive hormone secretion, preserving normal pituitary function whenever possible, reversing or treating impaired pituitary function and controlling the growth of the tumor and its mechanical effects on surrounding structures (Arafah & Nasrallah 2001).
In most pituitary adenomas, the primary treatment approach is transsphenoidal surgical adenomectomy. Generally, it is a very effective operation with low morbidity and mortality. However,
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a subsequent transsphenoidal approach may be needed to resect the residual suprasellar part of the tumor that descended after the first operation. A craniotomy may be needed in patients with a residual, suprasellar tumor that did not descend during transsphenoidal approaches. Repeated operations may also be needed if there is recurrence of the tumor. Radiation therapy is rarely recommended as the primary form of treatment, but it can be used as an adjunctive therapy in selected patients (Landolt 1999, Arafah & Nasrallah 2001, Sane in Välimäki et al. 2009 p.103). If hypopituitarism persists after surgery, it is managed by replacement of the deficient hormone(s) (Arafah & Nasrallah 2001, Melmed 2003).
There is also medical therapy available for the treatment of many types of pituitary adenomas. For example, the dopamine agonists bromocriptine and cabergoline reduce the size and hormonal hypersecretion of prolactinomas, and they are most often the primary treatment of prolactinomas. In acromegaly patients, the first-line pharmacological treatment is somatostatin analog (SSA) therapy.
SSAs are beneficial especially in patients with post-operative residual tumor activity, or in patients who are poor surgical candidates. Patients are most often treated with octreotide or lanreotide, which reduce plasma GH and IGF-I concentrations and in some cases also cause a moderate decrease in tumor size. In addition, a novel SSA, pasireotide, is currently in clinical trials. Also chimeric compounds with both somatostatin receptor and dopamine receptor affinity are being developed.
Novel drugs also include pegvisomant, a GH receptor antagonist that can be used for treatment of persistently high IGF-I levels and that can also be used in combination therapy with SSAs (Barkan et al. 1988, Molitch et al. 1997, Arafah & Nasrallah 2001, Manjila et al. 2010).
Recurrence of pituitary adenomas after apparently complete surgical resection is reported in 10-25%
of patients, usually within the first four years of operation. Therefore, periodic hormonal testing and repeated imaging studies are recommended to pituitary adenoma patients. It has further been recommended that follow-up of patients is maintained indefinitely (Arafah & Nasrallah 2001, Sane in Välimäki et al. 2009 p.126, 128).
Apart from clinically relevant adenomas that require effective treatment, some pituitary adenomas are found incidentally by radiological imaging of asymptomatic patients. These tumors are called incidentalomas. Their management is suggested to be based on periodic hormonal, clinical and radiological follow-up, particularly in cases having neither hormonal abnormalities nor clinical signs of the incidentaloma (Daly et al. 2007a).
4. Genetics of pituitary adenomas
Pituitary adenomas arise from the monoclonal expansion of a pituicyte that evades apoptosis and acquires unlimited replicative potential. Etiologic factors that have been implicated in pituitary tumorigenesis include genetic events, hormonal stimulation and growth factors. It is likely that all of these interact to initiate transformation and promote tumor-cell proliferation (Melmed 2003, Asa &
Ezzat 2009, Tanase et al. 2009). In the following sections, a number of genetic aberrations are described that are encountered either as somatic events in sporadic pituitary adenomas or as inherited defects in the context of familial susceptibility to pituitary tumors.
20 4.1 Sporadic pituitary adenomas
The vast majority of pituitary adenomas are sporadic. Their tumorigenesis has been studied with for example candidate gene approaches, genome-wide allelotyping and comparative genomic hybridization. The studies have identified LOH at putative tumor suppressor gene loci, putative markers of tumor progression and early alterations in tumors. Also hotspots that may indicate an unstable chromatin structure that is susceptible to deletions or epigenetic gene-silencing events and chromosomal aberrations have been discovered (Simpson et al. 2003, Pack et al. 2005). However, in many cases it is still unclear which of these alterations are involved in initiation and progression of sporadic pituitary oncogenesis.
4.1.1 GNAS/gsp oncogene
The guanine nucleotide-binding protein alpha stimulating activity polypeptide (GNAS) gene (20q13) encodes the guanosine nucleotide-binding protein Gs . It is required for the activation of adenylyl cyclase and subsequent generation of cyclic adenosine monophosphate (cAMP) in the cell. Acting as a cellular second messenger, cAMP binds to the cAMP-dependent protein kinase A (PKA) receptor and regulates a vast number of cellular processes, such as cell proliferation, differentiation and apoptosis. Activating mutations in GNAS cause increased activity of Gs , leading to increased cAMP levels (Vallar et al. 1987, Akintoye et al. 2002, Chin et al. 2002, Boikos & Stratakis 2007b). The term gsp oncogene has been assigned to these activating GNAS mutations due to their association with certain neoplasms. The gsp oncogene is found in 30-40% of GH-secreting adenomas, in a low percentage of NFPA and ACTH-secreting adenomas and in differentiated thyroid carcinomas. In addition, it is reported that Gs messenger ribonucleic acid (mRNA) levels can be high in some somatotropinomas without the gsp oncogene itself. The increased production of cAMP conferred by these mutations leads to overactivation of specific pathways involved in cell proliferation and specific programs of cell differentiation (Landis et al. 1989, Spada et al. 1998, Picard et al. 2007).
McCune-Albright syndrome (MAS) is a rare sporadic condition characterized by a triad of café-au-lait skin pigmentation, polyostotic fibrous dysplasia of the bone and hyperfunctioning endocrinopathies.
These include excess GH, hyperthyroidism and Cushing’s syndrome. The molecular etiology of this genetic but not inherited disease is an early embryonic postzygotic activating mutation of GNAS that results in constitutive Gs activation and elevated cAMP levels in the affected individual (Vallar et al.
1987, Akintoye et al. 2002, Boikos & Stratakis 2007b).
4.1.2 Other features of sporadic adenomas
Apart from the well-defined GNAS/gsp oncogene activation in sporadic pituitary adenomas, research of other pathways and factors is vigorously on-going. Putative mechanisms in sporadic pituitary tumorigenesis involve classic oncogenic signals, dysregulated growth factors and their receptors, epigenetically silenced tumor suppressor genes and chromatin remodeling (Asa & Ezzat 2009).
Examples of these mechanisms include activation of the proto-oncogene pituitary tumor transforming gene (PTTG), downregulation of the fibroblast growth factor receptor (FGFR) 2 or inactivation of RB1 (Woloschak et al. 1996, Abbass et al. 1997, Hunter et al. 2003). There are also other proteins
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emerging in recent studies, that may be involved in pituitary tumorigenesis, such as inhibitor of apoptosis stimulating protein of p53 (iASPP) and RWD-containing sumoylation enhancer (RSUME) (Fuertes et al. 2010, Pinto et al. 2010).
4.2 Familial pituitary adenomas
A minority (~5%) of pituitary adenomas occurs in a familial setting. The identification of genetic and molecular mechanisms underlying these conditions has greatly improved the understanding of them.
This process classically involves initial linkage analysis studies, the mapping and identification of relevant gene(s) and deciphering how abnormal protein expression leads to neoplastic changes at the molecular level (Daly et al. 2005, Tichomirowa et al. 2009). In the following sections, four familial conditions will be outlined where a known genetic defect leads to pituitary tumorigenesis. The two last sections will focus on familial pituitary adenomas with a yet unknown genetic background.
4.2.1 Multiple endocrine neoplasia type 1 (MEN1)
Multiple endocrine neoplasia type 1 (MEN1) (OMIM 131100) is an autosomal dominant disorder characterized by different combinations of tumors in the parathyroids, pancreas and the anterior pituitary. In addition, some patients may develop adrenal cortical tumors, gastrointestinal or thoracic neuroendocrine tumors, facial angiofibromas, collagenomas and lipomas. MEN1 arises from germline mutations in the MEN1 gene (11q13) (Chandrasekharappa et al. 1997, Elston et al.
2009, Thakker 2010, Table 3). Pituitary adenomas occur in about 30% of patients. Most often these are prolactinomas, although somatotropinomas, corticotropinomas or NFPAs are occasionally diagnosed as well (Trump et al. 1996, Tichomirowa et al. 2009, Thakker 2010). The prevalence of MEN1 has been estimated to be 0.02 – 0.2 in 1000 (Cazabat et al. 2009). MEN1 is characterized as familial if an affected individual has at least two of the three above-mentioned main MEN1 tumors and at least one first-degree relative has one of the three tumors (Marx et al. 1999, Brandi et al. 2001).
Although most MEN1 patients have inherited the disorder, molecular genetic studies have confirmed de novo mutations of the MEN1 gene in approximately 10% of patients with MEN1 (Lemos &
Thakker 2008). Approximately 10% of clinically suspected MEN1 patients do not have MEN1 mutations, suggesting that other predisposition genes may play a role in this phenotype (Hai et al.
2000; Daly et al. 2005).
The MEN1 gene consists of 10 exons encoding a 610 aa protein referred to as menin. It is a predominantly nuclear protein that has a role in transcriptional regulation, genome stability, cell division and proliferation. MEN1-associated tumors frequently exhibit LOH at the MEN1 locus, which is consistent with the tumor suppressor role of MEN1. Furthermore, although occasional somatic abnormalities of MEN1 have been reported in endocrine tumors, MEN1 is very rarely mutated in sporadic pituitary adenomas (Chandrasekharappa et al. 1997, Zhuang et al. 1997, Thakker 2010).
To date, over 1300 MEN1 mutations have been identified, of which the majority are predicted to lead to the truncation of menin. Interestingly, the phenotype of MEN1 is variable and shows an absence of phenotype-genotype correlations. Therefore, clinical features vary between patients of MEN1 families, even between identical twins (Bahn et al. 1986, Lemos & Thakker 2008, Thakker 2010).
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Table 3. Familial conditions with pituitary adenomas, data from Vierimaa et al. 2006, Agarwal et al.
2009, Tichomirowa et al. 2009, Gadelha & Frohman 2010, Kirschner 2010 and Thakker 2010.
Condition Predisposing gene (locus) Pituitary adenomas
MEN1 MEN1 (11q13) 30% of patients; mostly prolactinomas MEN4 CDKN1B (12p13) few patients discovered; some present
p15 (9p21), p18 (1p32), p21 (6p21)? with pituitary adenomas (GH, ACTH) CNC PRKAR1A (17q22-24) 10% GH-secreting adenomas ; 75%
unidentified (2p16) show GH/PRL overactivity
PAP AIP (11q13) low penetrance of pituitary adenomas;
mostly GH-secreting adenomas IFS AIP (11q13) in ~40%; unidentified acromegaly; gigantism
FIPA AIP (11q13) in ~15%; unidentified all pituitary tumor types; GH- and PRL-
secreting adenomas the most common
4.2.2 MEN4 (MEN1-like syndrome)
A recessively inherited MEN-like syndrome (MENX), causing multiple endocrine cancers including pituitary tumors, was first identified when occurring spontaneously in the rat. The MENX gene was later shown to be cyclin-dependent kinase n1b (cdkn1b) (Fritz et al. 2002, Piotrowska et al. 2004, Pellegata et al. 2006). In humans, the corresponding CDKN1B encodes the protein cyclin-dependent kinase inhibitor p27Kip1 on chromosome 12p13. The first heterozygous germline CDKN1B mutation (W76X) was found in a German family with acromegaly, primary hyperparathyroidism, renal angiomyolipoma and testicular cancer. Pedigree analysis revealed mutation segregation with the phenotype. Although the wildtype allele was retained in the tumor tissue, immunohistochemical staining of p27Kip1 showed no protein expression in the tumors. This supported an association between germline CDKN1B mutations and a heritable human MEN1-like condition called MEN4 (OMIM 610755) (Pellegata et al. 2006, Table 3). So far, only a handful of CDKN1B mutations have been found in suspected MEN1 cases with no MEN1 mutations (Georgitsi et al. 2007b, Agarwal et al.
2009). Interestingly, one of these (K25fs) was in a patient with an ACTH-secreting adenoma, but three others (P95S, -7G>C and X>Q) were in patients with no pituitary manifestation (Georgitsi et al.
2007b, Agarwal et al. 2009).
The CDKN1B gene consists of three exons encoding 198 aa. It is a well-established cyclin-dependent kinase inhibitor that negatively regulates cell cycle progression by inhibiting cyclin and cyclin- dependent kinase complexes in the nucleus (Pellegata et al. 2006, Lee & Kim 2009). It is reported that p27Kip1 is underexpressed or even absent in most pituitary adenomas (Lidhar et al. 1999). Intriguingly, the regulation of p27Kip1 expression involves both menin, and aryl hydrocarbon receptor interacting protein (AIP) through aryl hydrocarbon receptor (AHR) (Kolluri et al. 1999, Karnik et al. 2005, Milne et al. 2005, see section 5.2.1). The precise role and pathways of p27Kip1 in pituitary tumorigenesis are, however, yet to be elucidated. Interestingly, Besson et al. reported that independently of its role as a CDK inhibitor and tumor suppressor, p27Kip1 can act as an oncogene in vivo, promoting stem cell expansion and tumorigenesis in multiple tissues (Besson et al. 2007). In addition, there are reports of variations in other CDK inhibitor genes (p15, p18 and p21) that have been suggested to lead to a MEN1-like phenotype (Agarwal et al. 2009).
23 4.2.3 Carney complex (CNC)
Carney complex (CNC) (OMIM 160980) is a complex of myxomas, schwannomas, spotty skin pigmentation and endocrine overactivity (Carney et al. 1985, Tichomirowa et al. 2009, Table 3). It is a rare autosomal dominant disease that has been described in about 500 patients (Boikos & Stratakis 2007a). The median age at diagnosis is about 20 years, and the most common clinical manifestation at the time of presentation is spotty skin pigmentation. CNC patients have a decreased life-span, mostly due to heart-related causes such as cardiac myxomas (Stratakis et al. 2001). The main endocrine abnormalities seen in CNC are primary pigmented nodular adrenocortical disease, thyroid tumors and nodules, testicular tumors and acromegaly (Stergiopoulos & Stratakis 2003). Acromegaly occurs in roughly 10% of cases, but even 75% of patients have elevated GH, IGF-I or PRL levels or abnormal responses to dynamic pituitary testing (Pack et al. 2000, Stratakis et al. 2001). CNC-related acromegaly is distinguished by multifocal hyperplasia of mammosomatotropic cells that includes nonadenomatous pituitary tissue within the tumors (Kurtkaya-Yapicier et al. 2002).
Two candidate gene loci have been identified, one on chromosome 17q22-24 and the other on chromosome 2p16 (Stratakis et al. 1996, Casey et al. 1998). While no predisposing gene(s) have been found in the 2p16 locus, the 17q22-24 locus contains the gene encoding PKA regulatory subunit 1 alpha (PRKAR1A), which comprises 11 exons and encodes a protein of 381 aa. Mutations in PRKAR1A have been identified in up to 65% of CNC patients (Stratakis et al. 1996, Veugelers et al.
2004). PRKAR1A is a tumor suppressor gene, and most PRKAR1A mutations lead to mRNA instability, decreased or absent protein expression, and PRKAR1A haploinsufficiency in CNC tumors (Kirschner et al. 2000). LOH at 17q22-24 and allelic loss have been shown in CNC tumors.
PRKAR1A is the main component of PKA, which regulates most of the kinase activity catalyzed by the PKA holoenzyme in response to cAMP. This pathway is involved in the regulation of metabolism, cell proliferation, differentiation and apoptosis. The loss of PRKAR1A function enhances signaling through the PKA pathway. In the pituitary, the GHRH receptor uses the cAMP/PKA pathway to stimulate synthesis and the release of GH, suggesting that this could be one mechanism involved in the oncogenesis of somatotropinomas (Mayo et al. 1995, Kirschner et al. 2000, Groussin et al. 2002, Bossis & Stratakis 2004, Kirschner 2010).
4.2.4 Pituitary adenoma predisposition (PAP)
A fourth condition with familial pituitary adenomas, designated as pituitary adenoma predisposition (PAP) (OMIM 102200), was discovered by Vierimaa et al. in 2006 in three clusters of familial pituitary adenomas from Northern Finland. Two of the clusters could be linked by genealogy data.
The patients displayed low-penetrance susceptibility to somatotropinomas, prolactinomas and mixed adenomas. To identify the predisposing gene, whole-genome single nucleotide polymorphism (SNP) genotyping was performed. This was followed by linkage analysis, which provided evidence for linkage in 11q12-11q13, a region also previously implicated in isolated familial somatotropinoma (IFS) (Gadelha et al. 1999, Gadelha et al. 2000, Soares et al. 2005, Vierimaa et al. 2006).
The candidate locus was fine-mapped and the two pedigrees shared the linked haplotype, which segregated perfectly with somatotropinomas. Out of the 295 genes in the linked region, expression
